Diterpene cyclase and methods of use

ABSTRACT

An enzyme having diterpene cyclase activity has been purified from  P. elisabethae  using a series of chromatography steps. The purified enzyme has an apparent molecular weight of about 47 kilodaltons and an isoelectric point of about 5.1. The purifed enzyme catalyzed the cyclization of geranyl geranyl diphosphate to elisabethatriene.

CROSS REFERENCE TO RELATED APPLICATION

[0001] The present application claims the priority of U.S. provisionalapplication No. 60/351,984 filed Jan. 25, 2002.

STATEMENT AS TO FEDERALLY-SPONSORED RESEARCH

[0002] Not applicable.

FIELD OF THE INVENTION

[0003] The invention relates generally to the fields of biochemistry,enzymology, and marine biology. More particularly, the invention relatesto a purified enzyme useful for producing pseudopterosins.

BACKGROUND

[0004]Pseudopterogorgia elisabethae, a purple frilly seafan, is agorgonian commonly found in the shallow-water reefs of the tropicalAtlantic including regions of the Caribbean. P. elisabethae is ofparticular commercial importance as it has been found to containnumerous biologically active small molecule compounds. Among these,pseudopterosins (also known as terpenes, e.g., diterpenes) have beenshown to exhibit anti-inflammatory and analgesic properties, and arecurrently being used as topical agents in skin care products. In thebiosynthetic pathway shown in FIG. 1,pseudopterosin/seco-pseudopterosins are generated from geranyl geranyldiphosphate (GGPP). A key step in this pathway is the cyclization ofGGPP to elisabethatriene (compound 18). From elisabethatriene, theintermediate compounds 19-27 and pseudopterosin A are made. Theidentification of enzymes responsible for catalyzing key steps in thispathway, however, has been elusive. Identification of such an enzymewould facilitate the development of a chemoenzymatic method for theproduction of marine diterpenes.

SUMMARY

[0005] The invention relates to the purification and partial sequencingof an elisabethatriene cyclase from P. elisabethae. This cyclase isuseful for converting GGPP to elisabethatriene, a step involved in theproduction of seco-psuedopterosins, pseudotopterosins, and relatedmolecules. The purified enzyme is useful for synthesizing the foregoingmolecules. In addition, the purified cyclase should be useful for makingother diterpenes. The methods disclosed herein might also be used toproduce eleutherobin, an antimitotic agent isolated from the soft coralErythropodium caribaeorum.

[0006] Accordingly, the invention features a purified elisabethatrienecyclase such as a purified protein isolatable from a Pseudopterogorgiaelisabethae coral sample having an apparent molecular weight of about47,000 Da; an isoelectric point of about 5.1; and the ability to cyclizegeranyl geranyl diphosphate. The purified protein can be one thatincludes the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3 and/or SEQ ID NO:4. Fragments of the foregoing that are capable ofcatalyzing the formation of elisabethatriene from geranyl geranyldiphosphate are also featured in the invention.

[0007] In another aspect the invention features a method of purifying anelisabethatriene cyclase from a Pseudopterogorgia elisabethae sample.This method includes the steps of: (A) preparing a cell free extractfrom a Pseudopterogorgia elisabethae sample; (B) separating the cellfree extract into at least two fractions, one that exhibitselisabethatriene cyclase activity and one that does not; and (C)collecting the fraction that exhibits elisabethatriene cyclase activity.The step (A) of preparing a cell free extract from the Pseudopterogorgiaelisabethae sample can be performed by flash freezing thePseudopterogorgia elisabethae sample using liquid nitrogen; homogenizingthe frozen sample with a buffer and liquid nitrogen; separating thehomogenized sample into a cellular portion and a non-cellular portion;and collecting the non-cellular portion. The step (B) of separating thecell free extract can be performed by subjecting the cell free extractto one or more chromatographic separation steps such as DEAE ionexchange chromatography, phenyl sepharose chromatography, hydroxyapatitechromatography, and/or ion exchange chromatography with2-Propen-1-aminium, N,N,-dimethyl-N-2-propenyl-, chloride, polymer with1,4-bis(1-oxo-2-propenyl) piperazine and 2-methyl-2-propenamide.

[0008] The invention also provides a method for cyclizing geranylgeranyl diphosphate, e.g., to make elisabethatriene. This method isperformed by contacting geranyl geranyl diphosphate with a purifiedelisabethatriene cyclase under reaction conditions that result in theproduction of elisabethatriene. The elisabethatriene thus formed can beused as a substrate to produce other molecules involved inpseudopterosin synthesis. For example, elisabethatriene can be reactedto produce elisabethadione which can be reacted to produceelisabethadiol. The latter can be reacted to produce pseudopterosinagylcone which can be reacted to produce pseudopterosin A.

[0009] Unless otherwise defined, all technical terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Definitions of molecular biologyterms can be found, for example, in Rieger et al., Glossary of Genetics:Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991;and Lewin, Genes V, Oxford University Press: New York, 1994. Definitionsof organic chemistry and enzymology can be found, for example, in R. B.Silverman et al., The Organic Chemistry of Enzyme-Catalyzed Reactions,Academic Press: San Diego, Calif., 2000; and R. T. Morrisson et al.,Organic Chemistry, 6th edition, Addison-Wesley Publishing Co.: Boston,Mass., 1992.

[0010] As used herein, the terms “protein” and “polypeptide” are usedsynonymously to mean any peptide-linked chain of amino acids, regardlessof length or post-translational modification, e.g., glycosylation orphosphorylation. A “purified” polypeptide is one that has beensubstantially separated or isolated away from other polypeptides in acell, organism, or mixture in which the polypeptide occurs (e.g., 30,40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% free of contaminants).

[0011] A “purified elisabethatriene cyclase” is a purified proteinisolatable from P. elisabethae that has the ability to cyclize GGPP. Thephrase includes the purified native form of elisabethatriene cyclaseisolatable from P. elisabethae and having an apparent molecular weightof about 47,000 Da and an isoelectric point of about 5.1. It alsoincludes naturally occurring and non-naturally occurring proteins havinga similar structure (e.g., sharing 65, 70, 75, 80, 85, 90, 95, 97, 98,99% or more sequence identity) and enzymatic activity, e.g., allelicvariants of a native elisabethatriene cyclase, mutants of a nativeelisabethatriene cyclase, and forms of the enzyme produced byrecombinant DNA technology or chemical synthesis.

[0012] A “fragment” of an elisabethatriene cyclase polypeptide is aportion of an elisabethatriene cyclase polypeptide that is less thanfull-length (e.g., a polypeptide consisting of 5, 10, 15, 20, 30, 40,50, 75, 100 or more amino acids of native elisabethatriene cyclasepolypeptide), and preferably retains at least one functional activity ofnative elisabethatriene cyclase polypeptide (e.g., the ability tocyclize a GGPP substrate).

[0013] The term “antibody” includes polyclonal and monoclonal antibodiesas well as antibody fragments or portions of immunolglobulin moleculesthat can specifically bind the same antigen as the intact antibodymolecule.

[0014] As used herein, “bind,” “binds,” or “interacts with” means thatone molecule recognizes and adheres to a particular second molecule in asample, but does not substantially recognize or adhere to otherstructurally unrelated molecules in the sample. Generally, a firstmolecule that “specifically binds” a second molecule has a bindingaffinity greater than about 10⁵ to 10⁶ liters/mole for that secondmolecule.

[0015] Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. In thecase of conflict, the present specification, including definitions willcontrol. In addition, the particular embodiments discussed below areillustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The invention is pointed out with particularity in the appendedclaims. The above and further advantages of this invention may be betterunderstood by referring to the following description taken inconjunction with the accompanying drawings, in which:

[0017]FIG. 1 is a schematic overview ofpseudopterosin/seco-pseudopterosin biosynthesis pathways.

[0018]FIG. 2 is sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) analysis of chromatography fractions.Lane: 1) Low Molecular Weight Standard, 2) Cell-Free Extract, 3) IonExchange, 4) Dye Ligand, 5) Hydroxyapatite, 6) Purified Gel Slice.

[0019]FIG. 3 is a schematic overview of the chemical transformation ofelisabethatriene to elisabethadione.

[0020]FIG. 4 is a set of amino acid sequences (SEQ ID NOs:1-4)corresponding to elisabethatriene cyclase peptide fragments purifiedfrom P. elisabethae.

DETAILED DESCRIPTION

[0021] Elisabethatriene cyclase, an enzyme having diterpene cyclaseactivity, was purified from P. elisabethae by separating a cell freeextract of the coral using a series of chromatographic steps.Biochemical characterization of the purified protein indicated that ithad an apparent molecular weight of about 47,000 Da and an isoelectricpoint of about 5.1. The enzyme was partially sequenced. The purifiedenzyme retained its diterpene cyclase activity as evidenced by itsability to cyclize GGDP. This purified enzyme is thus useful in methodsof making elisabethatriene from a GGDP substrate and in methods forproducing diterpenes such as pseudopterosins, seco-pseudopterosins, andstructurally related molecules.

[0022] The below described preferred embodiments illustrate adaptationsof these compositions and methods. Nonetheless, from the description ofthese embodiments, other aspects of the invention can be made and/orpracticed based on the description provided below.

Biological Methods

[0023] Methods involving conventional biological techniques aredescribed herein. Such techniques are generally known in the art and aredescribed in detail in methodology treatises such as Molecular Cloning,3rd Edition, Sambrook and Russell, Cold Spring Harbor Press, 2001; andCurrent Protocols in Molecular Biology, ed. Ausubel et al., GreenePublishing and Wiley-Interscience, New York, 1992 (with periodicupdates). Methods in enzymology are discussed in Guide to ProteinPurification: Methods in Enzymology, Vol. 182, ed. M. P. Deutscher,Academic Press: San Diego, Calif., 1990.

Elisabethatriene Cyclase

[0024] The present invention provides a purified elisabethatrienecyclase polypeptide. A preferred form of the elisabethatriene cyclasefor use in the invention is the native form of the enzyme that can beisolated from P. elisabethae according to the methods described below.Biochemical characterization of the native form of elisabethatrienecyclase indicated that it has an apparent molecular weight of about 47kilodaltons and an isoelectric point of about 5.1. Partial amino acidsequencing indicated that this enzyme includes peptides having thesequences described herein as SEQ ID NOs:1-4. In addition to the nativeform, other variant forms of this enzyme are included within theinvention. Variants include fragments, analogs and derivatives of nativeelisabethatriene cyclase and may be made according to methods commonlyknown in the art. For example, such variants include a polypeptideencoded by a naturally occurring allelic variant of nativeelisabethatriene cyclase-encoding nucleotide sequence, a polypeptideencoded by a homolog of native elisabethatriene cyclase-encodingnucleotide sequence, and a polypeptide encoded by a non-naturallyoccurring variant of native elisabethatriene cyclase-encoding nucleotidesequence.

[0025] Elisabethatriene cyclase polypeptide variants have a peptidesequence that differs from native elisabethatriene cyclase polypeptidein one or more amino acids. The peptide sequence of such variants canfeature a deletion, addition, or substitution of one or more amino acidsof a native elisabethatriene cyclase polypeptide. Amino acid insertionsare preferably of about 1 to 4 contiguous amino acids, and deletions arepreferably of about 1 to 10 contiguous amino acids, and deletions arepreferably of about 1 to 10 contiguous amino acids. In someapplications, variant elisabethatriene cyclase polypeptidessubstantially maintain a native elisabethatriene cyclase polypeptidefunctional activity (e.g., cyclase activity). For other applications,variant elisabethatriene cyclase polypeptides lack or feature asignificant reduction in a elisabethatriene cyclase polypeptidefunctional activity. Where it is desired to retain a functional activityof native elisabethatriene cylcase, preferred elisabethatriene cyclasevariants can be made by expressing nucleic acid molecules within theinvention that feature silent or conservative changes. Variantelisabethatriene cyclase polypeptides with substantial changes infunctional activity can be made by expressing nucleic acid moleculesthat feature less than conservative changes.

[0026] Elisabethatriene cyclase polypeptide fragments corresponding toone or more particular motifs and/or domains or to arbitrary sizes, forexample, at least 5, 10, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300,350 and 400 amino acids in length are within the scope of the presentinvention. Isolated peptidyl portions of elisabethatriene cylcaseproteins can be obtained by screening peptides recombinantly producedfrom the corresponding fragment of the nucleic acid encoding suchpeptides. In addition, fragments can be chemically synthesized usingtechniques known in the art such as conventional Merrifield solid phasef-Moc or t-Boc chemistry. For example, an elisabethatriene cyclasepolypeptide of the present invention may be arbitrarily divided intofragments of desired length with no overlap of the fragments, orpreferably divided into overlapping fragments of a desired length. Thefragments can be produced (recombinantly or by chemical synthesis) andtested to identify those peptidyl fragments which can function as eitheragonists or antagonists of native elisabethatriene cyclase.

[0027] Another aspect of the present invention concerns recombinantforms of the elisabethatriene cyclase polypeptides. Recombinantpolypeptides preferred by the present invention, in addition to nativeelisabethatriene cyclase, are encoded by a nucleic acid that has atleast 85% sequence identity (e.g., 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100%) with a nucleic acid encoding nativeelisabethatriene cyclase. In a preferred embodiment, an elisabethatrienecyclase of the present invention is a coral elisabethatriene cyclase. Ina particularly preferred embodiment, an elisabethatriene cyclase has oneor more functional activities of native elisabethatriene cyclase.

[0028] Elisabethatriene cyclase variants can be generated throughvarious techniques known in the art. For example, elisabethatrienecyclase variants can be made by mutagenesis, such as by introducingdiscrete point mutation(s), or by truncation. Mutation can give rise toan elisabethatriene cyclase variant having substantially the same, ormerely a subset of the biological activity of native elisabethatrienecyclase. Other variants of elisabethatriene cyclase that can begenerated include those that are resistant to proteolytic cleavage.Whether a change in the amino acid sequence of a peptide results in anelisabethatriene cyclase variant having one or more functionalactivities of native elisabethatriene cyclase can be readily determinedby testing the variant for a native elisabethatriene cyclase functionalactivity in one or more of the assays described herein.

Purification of Elisabethatriene Cyclase

[0029] Elisabethatriene cyclase can be purified from a coral such as P.elisabethae by adapting a variety of known protein purificationtechniques. In the example described below, P. elisabethae coral sampleswere flash frozen using liquid nitrogen and then stored at −80° C. priorto use. Rapid freezing appeared to be important in maintaining theactivity of the enzyme. The frozen samples were homogenized andcentrifuged to remove insoluble debris. The resultant supernatant orcell free extract (CFE) was subjected to chromatographic separation.Those fractions containing diterpene cyclase activity were the fractionsthat contained the purified enzyme.

[0030] Diterpene cyclase activity can be assessed using any suitablesubstrate cyclization assay. For example, test samples/fractions can beincubated with radiolabeled (e.g., ³H) substrate (e.g., 1 μCi GGPP) at atemperature of about 27° C. for approximately 1-4 hours. The amount ofGGPP cyclized to elisabethatriene can then be determined.

[0031] Chromatographic separation of a CFE can be performed using avariety of known techniques in chromatography. For example, proteins inCFE can be separated according to molecular weight (e.g., using sizeexclusion chromatography) and/or charge (e.g., using ion exchangechromatography). In the method described below, a series ofchromatographic steps including DEAE ion-exchange chromatography, phenylsepharose chromatography, hydroxyapatite chromatography, and UNO™(Bio-Rad, Hercules, Calif.) chromatography was employed. Othertechniques may also be used to purify elisabethatriene cyclase. Forexample, in addition to conventional column chromatography,high-performance liquid chromatography (HPLC) and preparativeelectrophoresis might be used.

[0032] The isolation and purification methods described herein can beapplied to the isolation and purification of cyclases from organismsother than P. elisabethae (e.g., Erythropodium caribaeorum). Forexample, a purified E. caribaeorum cyclase would be useful for producingeleutherobin, an antimitotic agent isolated from E. caribaeorum.

Methods for Cyclizing A Substrate

[0033] The invention provides compositions and methods for cyclizing asubstrate (e.g., GGPP to elisabethatriene) using a purifiedelisabethatriene cyclase. A preferred substrate for cyclization is GGPP.Other potential substrates include GGPP analogues including 3-PhGGPP(see Mu et al., Bioorg. Med. Chem. 10:1207-1219, 2002; and Quellhorst etal., J. Biol. Chem. 276:40727-40733, 2001), farnesyl diphosphate (alsoknown as famesyl pyrophosphate, FPP), isomers of FPP (see Shao et al.,Org. Lett. 1:627-630, 1999), FPP analogues (see Micali et al.,Biochemistry 40:12254-12265, 2001; and Chehade et al., J. Am. Chem. Soc.124:8206-8219, 2002), as well as other phosphoisoprenoids (see Thoma etal., Biochemistry 39:12043-12052).

[0034] An example of a method for cyclizing GGPP to elisabethatrieneincludes the steps of providing purified elisabethatriene cyclase andcontacting the GGPP substrate with the purified elisabethatriene cyclaseunder reaction conditions that result in the production ofelisabethatriene. Any suitable reaction conditions that result in theproduction of elisabethatriene may be used. For example, ³H-GGPP (1 μCi)is incubated with a suitable amount of purified elisabethatriene cyclaseat 27° C. for 1-4 hours.

Pseudopterosin Biosynthesis

[0035] The invention provides methods for producing diterpenes, alsoknown as pseudopterosins. Such compounds have been isolated from marinecorals and many are useful as components in skin care products. OnceGGPP has been cyclized to produce elisabethatriene using the methodsdescribed herein, purified elisabethatriene can be used in methods toproduce pseudopterosins. For example, elisabethatriene can be aromatizedusing either Pd or a heteropoly acid in high yield (80-85%). Theresulting aromatic hydrocarbon could presumably be oxidized andsubsequently glycosylated using methods available in the literature togenerate a pseudopterosin-like molecule.

Anti-Elisabethatriene Cyclase Antibodies

[0036] Elisabethatriene cyclase polypeptides (or immunogenic fragmentsor analogs thereof) can be used to raise antibodies useful in theinvention. Such polypeptides can be produced by recombinant techniquesor synthesized as described above. In general, elisabethatriene cyclasepolypeptides can be coupled to a carrier protein, such as KLH, asdescribed in Ausubel et al., supra, mixed with an adjuvant, and injectedinto a host animal. Antibodies produced in that animal can then bepurified by peptide antigen affinity chromatography. In particular,various host animals can be immunized by injection with anelisabethatriene cyclase polypeptide or an antigenic fragment thereof.Commonly employed host animals include rabbits, mice, guinea pigs, andrats. Various adjuvants that can be used to increase the immunologicalresponse depend on the host species.

[0037] Antibodies within the invention therefore include polyclonalantibodies and, in addition, monoclonal antibodies (mAbs), single chainantibodies, Fab fragments, F(ab′)₂ fragments, and molecules producedusing a Fab expression library. Such antibodies can be of anyimmunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclassthereof. A hybridoma producing mAbs of the invention may be cultivatedin vitro or in vivo. The ability to produce high titers of mAbs in vivomakes this a particularly useful method of production.

[0038] Once produced, polyclonal or monoclonal antibodies can be testedfor specific elisabethatriene cyclase recognition by Western blot orimmunoprecipitation analysis by standard methods, for example, asdescribed in Ausubel et al., supra. Antibodies that specificallyrecognize and bind to elisabethatriene cyclase are useful in theinvention. For example, such antibodies can be used in an immunoassay tomonitor the level of elisabethatriene cyclase produced by a coral (e.g.,to determine the amount or subcellular location of elisabethatrienecyclase).

[0039] The antibodies of the invention can be used, for example, in thedetection of elisabethatriene cyclase in a biological sample. Antibodiesalso can be used in a screening assay to measure the effect of acandidate compound on expression or localization of elisabethatrienecyclase. Additionally, such antibodies can be used to interfere with theinteraction of elisabethatriene cyclase and other molecules that bindelisabethatriene cyclase.

[0040] The present invention is further illustrated by the followingspecific examples. The examples are provided for illustration only andare not to be construed as limiting the scope or content of theinvention in any way.

EXAMPLES Example 1

[0041] Isolation and Purification of Elisabethatriene Cyclase

[0042]P. elisabethae was collected from various sites in the Bahamas aswell as in the Florida Keys. The harvested coral was immediately flashfrozen in liquid nitrogen and stored at −80° C. A CFE was prepared byhomogenizing the flash frozen coral with phosphate buffer (pH 7.7, 5 mMβ-mercaptoethanol and 1 mM MgCl₂) and liquid nitrogen. The homogenatewas centrifuged at low speed to remove insoluble debris and thesupernatant used as the CFE.

[0043] Enzymatic activity in CFE preparations was typically assessed byincubating ³H-GGPP (1 μCi) with 30 mL of CFE at 27° C. for 1-4 hours.All CFE preparations analyzed showed the ability to catalyze thecyclization of GGPP to elisabethatriene. In other studies, CFE was shownto transform elisabethatriene to the pseudopterosins. Adding proteaseinhibitors in this process did not increase the enzymatic activity ofthe CFE.

[0044] To purify enzymatically active elisabethatriene cyclase, CFEsamples were subjected to DEAE ion-exchange chromatography, phenylsepharose chromatography, hydroxyapatite chromatography, and UNO™[2-Propen-1-aminium, N,N,-dimethyl-N-2-propenyl-, chloride, polymer with1,4-bis(1-oxo-2-propenyl)piperazine and 2-methyl-2-propenamide] ionexchange chromatography (Bio-Rad Laboratories, Hercules, Calif.). Activefractions were obtained in all cases. Fractions resulting from thesepurification techniques were concentrated by ultracentrifugation, andthen 0.5-100 μg (brought up to a total volume of 500 μl) was assayed forenzymatic activity by incubation with 1 μCi [1]³H-GGPP for 1 hour in 20mM Tris buffer (pH 7.7, 3 mM EDTA, 5 mM β-mercaptoethanol, 5 mM MgCl₂).Samples were quenched, extracted with 500 μl hexanes and partiallypurified by passing through a small silica gel pasteur pipette column.Elisabethatriene was then added as “cold carrier” and incubations werepurified by high-performance liquid chromatography (HPLC). During theHPLC analyses, fractions were collected before and after theelisabethatriene peak to demonstrate radiochemical purity and prove thatthe radioactivity obtained from the silica gel column was due toelisabethatriene. Radioactivity in HPLC fractions was quantified byscintillation counting.

[0045] As one example of a purification protocol used to purify thecyclase, a CFE sample was prepared using 140 g of flash frozen P.elisabethae and 200 mL of buffer A [20 mM Tris buffer (pH 7.7, 3 mMEDTA, 5 mM β-mercaptoethanol, 5 mM MgCl₂)]. This CFE was loaded onto aDEAE ion-exchange column and eluted with a step gradient of increasingNaCl from 0 to 10 mM in 5 mM increments using buffer B [20 mM Trisbuffer +1 M NaCl (pH 7.7, 3 mM EDTA, 5 mM β-mercaptoethanol, 5 mMMgCl₂)]. The active fractions, which eluted at 10 mM NaCl, wereconcentrated, exchanged by ultracentrifugation into buffer C [buffer A+1 M (NH₄)₂SO₄ (pH 7.7, 3 mM EDTA, 5 mM β-mercaptoethanol, 5 mM MgCl₂)],and subjected to chromatography on a phenyl sepharose CL-4B column.Proteins were eluted with a decreasing salt gradient from 15 to 0 mM(NH₄)₂SO₄ in 5 mM increments by increasing the percentage of buffer A.Most of the elisabethatriene cyclase activity eluted from the columnwith 0 mM (NH₄)₂SO4. The fractions obtained were concentrated, exchangedinto buffer D[1 mM potassium phosphate buffer (pH 6.0, 5 mMβ-mercaptoethanol, 5 mM MgCl₂)], and loaded onto a Bio-scale ceramichydroxyapatite CHT5-I column. Fractions were eluted from thehydroxyapatite column with a linear gradient of increasing potassiumphosphate concentration (35 to 110 mM) by increasing the percentage ofbuffer E[500 mM potassium phosphate buffer (pH 6.0, 5 mMβ-mercaptoethanol, 5 mM MgCl₂)]. Elisabethatriene cyclase activity wasobserved when the gradient reached approximately 100 mM potassiumphosphate. The final purification step included buffer exchange byultracentrifugation into buffer A and separation of protein on an UNO™Q1 continuous bed ion-exchange column. Protein was eluted using a lineargradient of increasing buffer B (40 to 140 mM NaCl) and theelisabethatriene activity eluted from the column between 50 and 80 mMNaCl.

[0046] After these purification steps, the purity of each proteinfraction was determined using SDS-PAGE electrophoresis (FIG. 2). Themolecular weight of the elisabethatriene cyclase was confirmed to beabout 47,000 daltons by comparison to calibration standards on aSephadex G-100 superfine size exclusion column. In addition, theisoelectric point (pI) of elisabethatriene cyclase was determined to be5.1 using an isoelectric focusing gel in which the bands were excisedand assayed for enzymatic activity. For sequencing, the SDS-PAGE bandsat about 47,000 daltons were excised from a 7.5% gel of active fractionsfrom the UNO™ ion-exchange column. The excised bands were subjected toEdman degradation sequencing. The sequences of four of the resultingpeptides sequences are shown in FIG. 4.

Example 2

[0047] Cyclizing GGPP to Elisabethatriene

[0048] Using elisabethatriene cyclase purified by column chromatographyas described in Example 1, GGPP was cyclized to elisabethatriene. Tocarry out the reaction, incubations were performed by incubating³H-GGPP(1 μCi) with purified elisabethatriene cyclase-containingfractions from chromatography columns at 27° C. for 1-4 hours. Purifiedcyclase elisabethatriene preparations analyzed catalyzed the cyclizationof GGPP to elisabethatriene. Adding protease inhibitors (e.g.,pepstatin, leupeptin and chymostatin) in this process did not increasethe enzymatic activity of the fractions as judged by the formation ofpseudopterosins.

Example 3

[0049] Pseudopterosin Biosynthesis

[0050] Pseudopterosin biosynthesis was characterized by performing adetailed chemical analysis of specimens of P. elisabethae collected indiverse geographic locations. P. elisabethae were collected from threeregions of the Bahamas and a recently discovered site in the FloridaKeys. From very careful analyses of these samples, over 20 metabolites,many of which are potential intermediates in thepseudopterosin/seco-pseudopterosin biosynthetic pathway (e.g., compounds18, 19, 20, 22, 24, 25, 27) were identified. Experiments were carriedout to confirm the intermediacy of a number of these metabolites. Theseexperiments suggested the pathway shown in FIG. 1 as the metabolicorigin of this group of diterpenes.

[0051] Using purified elisabethatriene cyclase coupled with other knownchemical methods, various pseudopterosins can be made from a GGPPsubstrate. Referring to FIGS. 1 and 3, the first step of the chemicaltransformation of elisabethatriene to elisabethadione is thearomatization of compound 18. Aromatization of compound 18 wasaccomplished using Pd/C in refluxing trigylme for 3 hours (GarrettTetrahedron Letters 3:191-194, 1969) which afforded compound 19 in ayield of ca. 50%. Alternatively, compound 19 can be generated usingH₅PMo₁₀V₂O₄₀(H₂O)₃₂ in 1,2-dichloroethane at 70° C. under an O₂atmosphere (R. Neuman et al., J. Org. Chem 54:4607-4610, 1989). Thesynthesis of compound 24 is completed by oxidation of compound 19 firstwith CF₃CO₃H/BF₃ and then NaIO₄ using standard methods (Ucciani et al.,J. Baudet Bull. Soc. Chim. Fr. 871, 1962). Compound 19 is oxidized tocompound 28 with dimethyldioxirane (DMD) in acidic medium. Bernini etal., Tetrahedron Lett. 41:1087-1090, 2000. Subsequently, oxidation ofcompound 28 is performed using Fremy's salt (potassiumnitrosodisulfonate) (Matsumoto et al., Biorg. Med. Chem. Lett.8:2945-2948, 1998) or nitrogen dioxide and oxygen (Bozell et al.,Tetrahedron Lett. 39:2261-2264, 1998).

Other Embodiments

[0052] It is to be understood that while the invention has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention, which is defined by the scope of the appended claims.Other aspects, advantages, and modifications are within the scope of thefollowing claims.

1 4 1 9 PRT Pseudopterogorgia elisabethae 1 Gly Gln Leu Asp Met His AspPro Ile 1 5 2 12 PRT Pseudopterogorgia elisabethae 2 Gly Tyr Pro Asn PhePro Ser Ile Ser Glu Met Lys 1 5 10 3 16 PRT Pseudopterogorgiaelisabethae MISC_FEATURE (7)..(7) X denotes an ambiguous residue 3 ArgAsp Glu Tyr Gly Asn Xaa Val Val Glu Thr Phe Val Glu Asn Leu 1 5 10 15 414 PRT Pseudopterogorgia elisabethae 4 Gly Leu Leu Asp Ala Leu Gln GlyIle Val Asp Gly Arg Asp 1 5 10

What is claimed is:
 1. Purified elisabethatriene cyclase.
 2. A purifiedprotein isolatable from a coral sample comprising Pseudopterogorgiaelisabethae or a purified fragment of the protein capable of catalyzingthe formation of elisabethatriene from geranyl geranyl diphosphate, thepurified protein having the following characteristics: (A) an apparentmolecular weight of about 47,000 Da; (B) an isoelectric point of about5.1; and (C) the ability to cyclize geranyl geranyl diphosphate.
 3. Thepurified protein of claim 2, wherein the purified protein comprises anamino acid sequence selected from the group consisting of: SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.
 4. The purified protein ofclaim 3, wherein the purified protein comprises the amino acid sequencesof SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.
 5. A method ofpurifying an elisabethatriene cyclase from a Pseudopterogorgiaelisabethae sample, the method comprising the steps of: (A) preparing acell free extract from a Pseudopterogorgia elisabethae sample; (B)separating the cell free extract into at least one fraction thatexhibits elisabethatriene cyclase activity and at least one fractionthat does not exhibit elisabethatriene cyclase activity; and (C)collecting the at least one fraction that exhibits elisabethatrienecyclase activity.
 6. The method of claim 5, wherein the step (A) ofpreparing a cell free extract from the Pseudopterogorgia elisabethaesample comprises: flash freezing the Pseudopterogorgia elisabethaesample using liquid nitrogen; homogenizing the frozen sample with abuffer and liquid nitrogen; separating the homogenized sample into acellular portion and a non-cellular portion; and collecting thenon-cellular portion.
 7. The method of claim 6, wherein the step (B) ofseparating the cell free extract comprises subjecting the cell freeextract to at least one chromatographic separation step.
 8. The methodof claim 7, wherein the chromatographic separation step comprises DEAEion exchange chromatography.
 9. The method of claim 7, wherein thechromatographic separation step comprises phenyl sepharosechromatography.
 10. The method of claim 7, wherein the chromatographicseparation step comprises hydroxyapatite chromatography.
 11. The methodof claim 7, wherein the chromatographic separation step comprises ionexchange chromatography with 2-Propen-1-aminium,N,N,-dimethyl-N-2-propenyl-, chloride, polymer with1,4-bis(1-oxo-2-propenyl)piperazine and 2-methyl-2-propenamide.
 12. Amethod comprising the step of contacting geranyl geranyl diphosphatewith the purified protein or fragment of the protein of claim 2 underreaction conditions that result in the production of elisabethatriene.13. The method of claim 12, further comprising reacting theelisabethatriene to produce elisabethadione.
 14. The method of claim 13,further comprising reacting the elisabethadione to produceelisabethadiol.
 15. The method of claim 14, further comprising reactingthe elisabethadiol to produce pseudopterosin agylcone.
 16. The method ofclaim 15, further comprising reacting the pseudopterosin agylcone toproduce pseudopterosin A.